Exploring L-tyrosine and L-DOPA biosynthesis in faba bean ( Vicia faba L.)

BACKGROUND AND AIMS: L-DOPA is an important pharmaceutical that accumulates to high levels in the legume faba bean (Vicia faba). L-DOPA is likely derived from L-tyrosine but the responsible enzyme (L-tyrosine oxidase) remains unknown. Availability of L-tyrosine may be a key factor controlling L-DOPA accumulation. In legumes, L-tyrosine is supplied via either a plastidial TyrA enzyme (ADH) or a deregulated cytosolic homolog (PDH). This study aimed at identifying L-tyrosine oxidase and TyrA genes from faba bean. METHODS: We used gene-to-metabolite correlations and homology-based searches to select fifteen L-tyrosine oxidase candidates, which were tested in yeast and in the model plant Nicotiana benthamiana. We also used isotopically labeled L-tyrosine to measure biosynthetic activity in different faba bean tissues and to test an alternative biosynthetic hypothesis. Three putative faba bean TyrA genes were inferred by homology and assayed in N. benthamiana by co-expression with a known L-tyrosine oxidase, CYP76AD6. KEY RESULTS: None of the L-tyrosine oxidase candidates produced L-DOPA upon heterologous expression. Feeding experiments showed a lack of correlation between L-DOPA accumulation and biosynthetic capacity. Feeding studies also refuted an alternative route to L-DOPA by oxidation of 4-hydroxyphenylpyruvate. Of the TyrA genes, two were able to increase L-tyrosine levels in N. benthamiana 2-3-fold (VfADH and VfPDH), and one of them was able to boost the levels of L-DOPA derivatives up to 6-fold (VfADH). CONCLUSIONS: The faba bean L-tyrosine oxidase remains unidentified, with a hypothetical transport of L-DOPA across tissues possibly having confounded our correlation-based selection strategies. In N. benthamiana, both VfADH and VfPDH can increase the levels of L-tyrosine, while VfADH can further boost the levels of L-DOPA derivatives. Our work delivers a strategy to boost the provision of L-tyrosine in N. benthamiana and provides valuable insights in the search for the elusive L-tyrosine oxidase from faba bean.